Determination of 17-Hydroxy-11-dehydrocorticosterone with Anthrone Reagent JOSEPH J . HOLECHEK
AND
A N N E R. COLLINS
Research U n i t , National Cancer I n s t i t u t e , United States Public Health Seruice Hospital, Baltimore, M d . HE cuiient interest in cortisone and related compounds haci rincreaced the need for accurate means of assay. Kumerous mrthods are available for the qualitative and quantitative estimation of iteroids and various constituent functional groups on the St(3roid molecule (1,4-8).
250
a 4
si.’ectrophotonieter. I t is necessary to allow the reaction to continue for 1 hour to ensure optimum color development. The color produced is stable a t least 1 hour after development. The measurements are made against a blank, consisting of 2 nil. of absolute ethyl alcohol and 2 ml. of anthrone reagent, which has been treated in the same manner as the steroid solution.
l
DISCUSSION
The spectral absorption curves which are produced by the interaction of anthrone with cortisone acetate or the free alcohol show maxima a t 415, 480, 590, and 635 mp (Figure 1). Spectrophotometric measurements are made at 480 mp. -4t this wave length the optical density per unit concentration is greater than a t either 590 or I335 mp. Anthrone itself absorbs considerably a t 415 mp and would thus interfere with the accurate measurement of the chromogen produced by the interaction of anthrone with cortisone a t this wave length. Since the composition of the cortisone-anthrone chromogen is not knoxn, apparent molecular extinction coefficients from the spectral absorption curves shown in Figure 1 have been calculated on the basis of the equivalent weight of the steroid used to allow a coniparison of the optical densities of the chromogens pi oduced bv cortisone acetate and cortisone. Although the two compounds have comparahle ipectral absorption maxima, the free alcohol gives slightly higher optical density values at these wave lengths.
4
150r
r
90-
440
400
480
520
560
600
640
1.4
WAVELENGTH (rnp) Figure 1.
I
Spectral Absorption Curves of Chromogens Produced
Cortisone (1.81 mg.) in reaction with: 0, anthrone reagent; 1, sulfuric acid Cortisone acetate (1.64 mg.) in reaction with: 0 , anthrone reagent: sulfuric acid. Length of cell, 1 om.
m,
The effect of the anthrone reagent on various steroid\ (3) and the use of the anthrone reaction as a means of measuring dehydroisoandrosterone and total 17-ketosteroids has been described ( 2 ) . Graff and associates (5)have shown that cortisone produces chromogens when allo~-edto react with the anthrone reagent. T o chromogen formation was obtained u hen using sulfuric acid alone with cortisone. The production of color when cortisone i a treated with the anthrone reagent is the hasis foi the method piewnted here.
g .4t
c
PROCEDURE
.2L&!L
Reagents. Absolute ethyl alcohol, 99.9Yc Anthrone reagent, 0.27, anthrone (9,10-dihydro-9-keto:mthracene) in 9Sc;C sulfuric acid (c.P.; sp. gr., 1.83). Method. The steroid to be investigated is dissolved in absolute ethyl alcohol. A suitable concentration of the steroid was found to be approximately 1 mg. per milliliter. Aliquots of the steroid solution are transferred to test tubes of uniform size, (e. g., 19 X 150 mm.) and brought up to a volume of 2 ml. with absolute ethyl alcohol. A 2-ml. portion of anthrone reagent is added in such a manner as to form a layer under the alcoholic steroid solution. Mixing is obtained by means of a glass rod having a flattened end. Care must be taken in mixing the lagers to prevent vigorous reaction and subsequent boiling away of the alcohol. The mixture is allowed to react for 1 hour, after which aliquots are transferred to 1-cm. spectrophotometer cells, and the optical density is measured a t 480 mp on a Beckman model DU
= OO
0.4
0.8
1.2
I.6
2.0
CONCENTRATION (rng.1 ml.)
Figure 2. Standardization Curves for Cortisone and Cortisone Acetate i n Reaction with the Anthrone Reagent The color produced by the reaction of anthrone with either cortisone acetate or the free alcohol follows the Beer-Lambert law over a convenient range of concentration (0.5 to 2.0 nig. per milliliter), as shown in Figure 2.
99 1
ANALYTICAL CHEMISTRY
992 SUMMARY
A colorimetric method is described for the estimation of cortisone employing the interaction of the steroid with the anthrone reagent. Concentrations varying from 0.5 to 2.0 mg. per milliliter of either cortisone or cortisone acetate may he quantitatively determined by the method described.
LITERATIJRE CITED (1) (2)
187, 741 (1952).
(3) Graff, 31. M., AIcElroy, 3.T., and AIooney, A . L., Ibid., 195, 351 (1952). (4)
ACKNOWLEDGMENT
The authors are indebted to Morris >I. Graff for his helpful interest in the preparation of this manuscript. The authors are grateful to Elmer illpert of JIerck and Co., Inc., for supplying samples of cortisone and cortisone acetate used in this work.
Daughaday, W. H., Jaffe, H., and Williams, R. H., J . Clin. Endocrinol., 8, 166 (1948). Graff, hf. M., Holechek, J. J., and Collins, A. R., J . Biol. Chern.,
Lowenstein, B. E., Corcoran, A. C., and Page, I. H., Endocrinology, 39, 82 (1946).
( 5 ) IIader, W.J., and Buck, R. R., Ax.4~.CHEaf., 24, 666 (1952). (6) Porter, C. C., and Silber, R. H., J . Bid. Chem.. 185, 201 (1950). (7) Talbot, iK.B., Saltaman, A. H., Wixom, R. L.. and Wolfe, J. K., Ibid., 160, 535 (1945). (S) T'enning, E. H., Hoffman, 31. AI., and B!.owne, J. S. L., E I L ~ O crinology, 35, 49 (1944). RECEIVED for review December 3 , 1952.
Accepted March 6, 1953.
Spectrophotometric Determination of Thorium in Black Sands CHARLES V. BANKS, DONK W. KLINGMilN, AXD ChROL H. BYRD I n s t i t u t e f o r A t o m i c Research and Department of Chemistry, lowa State College, A m e s , Zowa YCREASED
interest in thorium chemistry during the past few
1-years has created the demand for a relatively simple quantitative method for determining small amounts of thorium in sands
and ores. The best source of this element is monazite sands, which contain 3 to 15% thoria. -4method for the analysis of monazite sands has been suggested by Banks and Bgrd (a), and in that paper it was noted that the method was not applicable to black sands without some modification. Black sands are a comples mixture containing many other minerals in addition to monazite sands; thus, their thoria contents are in the lower range of 0.1 to 1.0%. It is the purpose of this paper to outline a procedure for extending this method to these more complicated ores.
The thorium is stripped from the solvent phase by adding 20 nil. of water and again shaking for 20 seconds. The aqueous phase is drained into a 100-ml. volumetric flask; the water strip is repeated once more. The contents of the flask are diluted to volume. The water strip is frequently observed to have a faint yellow calor, probably due to the osidation products of mesityl oxide. This oxidation can he minimized b y avoiding the use of warm solutions during the estractions and working with maximum efficienc.v.
Table I. Analysis of Black Sands % Tho? Reporteda
% Tho, Found
0 33 0.25 0 089 0.163 0.53
0 . 3 2 i0 . 0 1 0.22 =t0 . 0 1 0 . 0 9 6 =t0 . 0 1 0 . 1 4 & 0 01
MATERIALS AND APPARATUS
The materials and apparatus used in this procedure are the same as those used by Banks and Byrd ( 2 ) . PROCEDURE
Preparation and Decomposition. The samples are ground to 200 mesh with a mortar and pestle and mixed t,hroughly to ensure homogeneity. It is important that the particle size be small in order to facilitate complete fusion as rapidly as possible. An approximately 0.2-gram sample is weighed, placed in a 50-nil. platinum crucible, and fused according to the niethod for monazite sands ( 2 ) . The cooled melt' is transferred to a 125-rn1. platinum dish, using 50 ml. of hot water. If difficult'y is experienced in complete transfer, a few milliliters of dilute hydrofluoric acid will facilitate the operation. Twenty milliliters of 48% hydrofluoric acid are added, and the mixture is digested under infrared heating lamps (or on the steam bath) for 30 minutes. The melt is pulverized and stirred occasionally during the digestion. Separation and Solution of the Fluorides. The hot mixture is transferred to a 50-nil. Lusteroid centrifuge tube and separated a t the full speed of the centrifuge for 3 to 5 minutes. The supernatant liquid is poured off carefully, and the residue is washed twice with 10 to 15 nil. of hot 16% hydrofluoric acid. (Caut,ion: The separation must be carried out in a well-ventilated hood.) This further facilitates the volatilization of any remaining silica. .1 hydrofluoric acid wash bottle can be construct'ed from a polyethylene bottle using a Teflon tubing tip. These fluorides are transferred with a minimum amount of hot water to a 150-ml. beaker containing 19 grams of aluminum nitrate 9-hydrate and 2.5 ml. of concentrated nitric acid. The solution is warmed on the hot plate to dissolve the fluorides as the fluoaluminates, evaporated to 20 ml., and allowed to cool. Extraction with Mesityl Oxide. The first part of the estraction is carried out according to the procedure of Banks and Byrd (2). If .a white suspension should form a t the interface of the aqueous and solvent phase during the extraction, it should be kept with the solvent phase. The nature of this suspension is not understood, but it usually forms during the extraction and the first scrub. I t tends to separate on standing but the extraction should be carried out as rapidly as possihle since nitric acid sle~v1y oxidizes mesityl oxide, turning it brorn.
0 . 4 8 =s 0 . 0 1
Difference 0.01 0.03 0.01 0.02
0.05 United States Bureau of Mines value obtained by the niethod of &onstadt and Eberle (3). a
Spectrophotometric Determination. The aliquot take11 tor analysis should contain about 70 micrograms of thoria, since the Beckman Model DC spectrophotometer is most accurate for an absorbance of about 0.45 (1). The aliquot is pipetted into a 100ml. beaker containing 10 ml. of 0.02% thorin and is diluted to about 40 ml. The pH is adjusted with a pH meter to about 0.92 using perchloric acid. The solution is transferred to a 50-ml. volumetric flask and diluted to volume; after thorough mixing it iq ready for measurement. The reference blank is a solution containing the same concentration of reagent and a t the same pH as the sample. The ahsortlance is measured on a Beckman Model DU spectrophotometer a t a wave length of 545 mH and a slit width of 0.17 mm. using 5-cm. Corex cells. Solutions should be read within 4 hours ai ter being prepared. Calculations. This system conforms to Beer's law a t least up to a concentration of 100 micrograms of thoria per 50 ml. Since the reagent thorin is available in varying degrees of purity the analyst should construct a Beer's law plot and determine the factor for converting absorbance to thoria concentration for this reagent. This can be accomplished by preparing a series of solutions containing known concentrations of thoria and plotting the concentrations against the absorbance. If the absorbance is plotted as the ordinate, and micrograms of thoria per 50 ml. as the abscissa, the best straight line connecting these points will have a slope equal to the reciprocal of the conversion factoi, RESCLTS
The black sands samples used in this study were obtained from Reuben Kronstadt of the Cnited States Bureau of Mines, Raleigh, S. C. Table I lists the values obtained in this laboratory and by the Gnited States Bureau of Mines.
